Summary The need to identify chemicals for their potential to cause reproductive and developmental (R/D) toxicity (R/D) is a significant public health challenge for toxicologists. In vivo testing for reproductive and developmental toxicity assessment uses a large number of animals, yet these studies are criticized for their inefficiency. The National Research Council (NRC)?s report ?Toxicity Testing in the 21st Century? envisioned that in vivo animal testing will eventually be replaced by a combination of in silico and in vitro approaches. Therefore, the need to identify in vitro systems for reproductive and developmental toxicity testing has grown. ReproTOX has established an animal-free testicular cell co-culture system (Mini-Testis) from testicular cell lines. Both morphology and cellular biomarkers confirmed that this animal-free 3D-co-culture model support in vitro spermatogenesis. Our 32-compound preliminary study revealed a stronger correlation of IC50 from the Mini-Testis model with in vivo reproductive toxicity as compared to any single testicular cell culture model. These initial results suggest that our Mini-Testis model might be a valuable screening tool for reproductive toxicants and prioritizing chemicals for further testing. The specific aims of this proposal, therefore, are to (1) establish a toxicity-based high throughput Mini-Testis model for R/D toxicity evaluation and (2) develop an integrated pathway-based high content (HCA) and high throughput (HT) screening assay in the Mini-Testis model for R/D toxicity evaluation. The project outcome will be a cost-efficient, pathway-based in vitro Mini- Testis model for potential R/D toxicity screening of chemicals.